Project Summary/Abstract Polyketide natural products are widespread across all domains of life, occurring predominantly in bacteria, plants, and fungi. Their vast structural diversity has enabled nearly 50 years of research into a large, and growing, repertoire of polyketide-type compounds in search for potentially useful bioactivities. Indeed, many polyketides have been identified which exhibit antibacterial, anticancer, antifungal, antiviral, anti-inflammatory, immunosuppressive, and cholesterol-lowering properties; thus, underscoring their value as a source of potential drugs. Conveniently, a subset of polyketide synthases (PKSs), the enzymes that forge polyketides in nature, resemble modular assembly lines with multiple catalytic domains contained on a single polypeptide. In this way the polyketide product structures are templated according to the observed PKS domain order. Thus, there appears to exist a natural program underpinning the biosynthetic logic of polyketides, leading many to wonder whether PKSs can be reprogrammed to endow them with unnatural functions. The modular PKSs in bacteria (accounting for ~50% of polyketides) offer exciting prospects for combinatorial engineering of unnatural PKSs with novel function, and many efforts have attempted to create such PKSs by substituting catalytic domains from exogenous PKS sources. However, the catalytic efficiencies of many of these hybrid enzymes are compromised for reasons that are not well understood. Reliable and successful reprogrammability of PKSs therefore requires a thorough understanding of the structures and mechanisms governing natural enzyme function. We first aim to uncover the molecular bases of acyl carrier protein (ACP) recognition by two core PKS catalytic domains, the ketosynthase (KS) and acyltransferase (AT), from a bacterial modular PKS. This goal has been challenged by the transient and reactive nature of ACP/catalytic-domain complexes during polyketide processing. To mitigate this roadblock, we plan to incorporate unnatural amino acids (UAAs) with electrophilic functional groups into the ACPs for interdomain crosslinking with reciprocal Cys/Lys nucleophiles. Identified crosslinked species will be applied to structural and empirical modeling studies. A second Aim, aided by high- affinity antigen-binding fragments (Fabs), seeks to understand the structure-function relationships of a PKS ketoreductase, and new, supporting structural data are reported here. In the last Aim, we propose to study the conformational dynamics of a PKS module using Fabs, chemical crosslinking, and other stabilizers combined with single particle cryo-electron microscopy (cryo-EM) to obtain a high-resolution structure. Preliminary electron microscopy and activity data relevant to this Aim are reported, and strategies to improve the particle quality are outlined. Finally, the proposed research is expected to offer unique training opportunities in the areas of UAA incorporation and single particle cryo-EM; the latter of which is facilitated by collaboration with Prof. Wah Chiu?s lab (Stanford/SLAC) and close proximity to the Stanford/SLAC Cryo-EM Center (S2C2).